Project Summary/Abstract: During meiosis, a diploid cell undergoes one round of replication followed by two rounds of chromosome segregation to ultimately produce haploid gametes. A failure to properly segregate chromosomes in meiosis can result in infertility, miscarriage, and trisomy conditions, such as Down syndrome. Despite the importance of meiosis, there is a lack of molecular understanding of how cell-cycle regulatory networks function to ensure that chromosomes properly attach to spindle microtubules in meiosis I and meiosis II. The objective of this proposal is to determine the mechanisms of meiotic regulation that ensure proper chromosome segregation in meiosis. These studies employ S. cerevisiae as the model organism due to the ease in developing tools to address mechanistic questions. These innovative tools will allow the investigation of how cells correct improper microtubule-kinetochore attachments, how cells set the duration of meiosis, and how crossover position along homologous chromosomes affect microtubule-kinetochore attachments. The rationale for the proposed research is that the questions were chosen to focus on processes that are likely to be highly conserved, allowing the findings in budding yeast to uncover general mechanisms of meiotic regulation. Strong preliminary data guided the following three specific aims: 1) Investigate how spindle checkpoint proteins crosstalk with kinases and phosphatases at the kinetochore to regulate the timing and fidelity of chromosome segregation in meiosis; 2) Determine how the spindle checkpoint is prematurely silenced during meiosis when chromosomes are not correctly attached to spindle microtubules; and, 3) Determine how crossover location along a chromosome can affect the fidelity of kinetochore-microtubule attachments. In the first aim, cell-cycle regulators at the kinetochore will be tested for their role in maintaining the timing and accuracy of meiosis. The second aim tests the novel hypothesis of a meiosis-specific mechanism for silencing the spindle checkpoint to ensure the formation of gametes, even without proper chromosome segregation. The third aim addresses the long unanswered question of why chromosomes with crossovers at sub-optimal positions are more likely to mis-segregate. Strains will be developed that have chromosomes engineered to form a crossover at a specific site and fast live cell imaging will monitor the attachment to microtubules. The innovative approach of combining the latest imaging technologies to monitor kinetochore-microtubule attachments in engineered strains allows the testing of novel hypotheses about cell-cycle regulation. The proposed research is significant because the results are expected to reveal general principles of meiotic regulation important for protecting genome integrity. Ultimately, the results will further our understanding of how errors in meiosis facilitate developmental abnormalities.